Bilirubin is a product of hemoglobin turnover that is poorly soluble in water and is therefore largely associated with albumin in plasma. A small fraction of the total plasma bilirubin however is soluble in the aqueous phase. This unbound or free fraction is able to permeate the blood brain barrier and, at elevated levels, is neurotoxic [Ahlfors C E, Wennberg R P, Ostrow J D and Tiribelli C. Unbound (free) bilirubin: improving the paradigm for evaluating neonatal jaundice. Clin Chem 55: 1288-1299, 2009]. Under normal conditions total serum bilirubin is maintained at low levels by a regulated balance between production and excretion of bilirubin. However in newborns the mechanisms of regulation may not be sufficiently matured so that the production-excretion balance often favors accumulation, giving rise to the yellow color of jaundice in about 60% of newborns [Maisels M J and McDonagh A F. Phototherapy for neonatal jaundice. N Engl J Med 358: 920-928, 2008]. In most cases this imbalance is benign or may in fact be beneficial and for most newborns resolves spontaneously [Wennberg R P, Ahlfors C E, Bhutani V K, Johnson L H and Shapiro S M. Toward understanding kernicterus: a challenge to improve the management of jaundiced newborns. Pediatrics 117: 474-485, 2006; Gopinathan V, Miller N J, Milner A D and Rice-Evans C A. Bilirubin and ascorbate antioxidant activity in neonatal plasma. FEBS Lett 349: 197-200, 1994]. Concentrations of unbound bilirubin can however rise to levels that are neurotoxic, resulting in deficits ranging from reversible hearing defects to the more severe neurological sequelae of kernicterus that in rare instances include death [Ahlfors C E, Wennberg R P, Ostrow J D and Tiribelli C. Unbound (free) bilirubin: improving the paradigm for evaluating neonatal jaundice. Clin Chem 55: 1288-1299, 2009].
Early intervention using phototherapy or exchange transfusion can treat bilirubin mediated neurotoxicity in neonates [Maisels M J and McDonagh A F. Phototherapy for neonatal jaundice. N Engl J Med 358: 920-928, 2008; Morris B H, Oh W, Tyson J E, Stevenson D K, Phelps D L, O'Shea T M, McDavid G E, Perritt R L, Van Meurs K P, Vohr B R, Grisby C, Yao Q, Pedroza C, Das A, Poole W K, Carlo W A, Duara S, Laptook A R, Salhab W A, Shankaran S, Poindexter B B, Fanaroff A A, Walsh M C, Rasmussen M R, Stoll B J, Cotten C M, Donovan E F, Ehrenkranz R A, Guillet R and Higgins R D. Aggressive vs. conservative phototherapy for infants with extremely low birth weight. N Engl J Med 359: 1885-1896, 2008; Kuzniewicz M W, Escobar G J and Newman T B Impact of universal bilirubin screening on severe hyperbilirubinemia and phototherapy use. Pediatrics 124: 1031-1039, 2009]. Guidelines for intervention depend principally on total bilirubin levels, with account taken for gestational age and risk factors [Bhutani V K, Johnson L and Sivieri E M. Predictive ability of a predischarge hour-specific serum bilirubin for subsequent significant hyperbilirubinemia in healthy term and near-term newborns. Pediatrics 103: 6-14, 1999]. However fundamental biochemical and increasing clinical evidence predicts that unbound bilirubin rather than total bilirubin should more accurately correlate with bilirubin mediated neurotoxicity [Ahlfors C E, Wennberg R P, Ostrow J D and Tiribelli C. Unbound (free) bilirubin: improving the paradigm for evaluating neonatal jaundice. Clin Chem 55: 1288-1299, 2009; Wennberg R P, Ahlfors C E and Aravkin A Y. Intervention guidelines for neonatal hyperbilirubinemia: an evidence based quagmire. Curr Pharm Des 15: 2939-2945, 2009; Ahlfors C E, Amin S B and Parker A E. Unbound bilirubin predicts abnormal automated auditory brainstem response in a diverse newborn population. J Perinatol 29: 305-309, 2009; Oh W, Stevenson D K, Tyson J E, Morris B H, Ahlfors C E, Bender G J, Wong R J, Perritt R, Vohr B R, Van Meurs K P, Vreman H J, Das A, Phelps D L, O'Shea T M and Higgins R D. Influence of clinical status on the association between plasma total and unbound bilirubin and death or adverse neurodevelopmental outcomes in extremely low birth weight infants. Acta Paediatr 99: 673-678, 2010]. Therefore unbound bilirubin should be superior to total bilirubin for identifying neonates at risk for bilirubin neurotoxicity [Ahlfors C E. Predicting bilirubin neurotoxicity in jaundiced newborns. Curr Opin Pediatr 22: 129-133, 2010].
Aggressive phototherapy in premature infants is designed to maintain total bilirubin below 5 mg/dL [Morris B H, Oh W, Tyson J E, Stevenson D K, Phelps D L, O'Shea T M, McDavid G E, Perritt R L, Van Meurs K P, Vohr B R, Grisby C, Yao Q, Pedroza C, Das A, Poole W K, Carlo W A, Duara S, Laptook A R, Salhab W A, Shankaran S, Poindexter B B, Fanaroff A A, Walsh M C, Rasmussen M R, Stoll B J, Cotten C M, Donovan E F, Ehrenkranz R A, Guillet R and Higgins R D. Aggressive vs. conservative phototherapy for infants with extremely low birth weight. N Engl J Med 359: 1885-1896, 2008]. However Morris et al found no difference in outcome (death and neurologic development impairment) for patients treated to maintain total bilirubin at less than 5 mg/dL and those maintained at less than 8 mg/dL. However a follow up study by these investigators found that outcomes were well correlated with unbound bilirubin but not total bilirubin [Oh W, Stevenson D K, Tyson J E, Morris B H, Ahlfors C E, Bender G J, Wong R J, Perritt R, Vohr B R, Van Meurs K P, Vreman H J, Das A, Phelps D L, O'Shea T M and Higgins R D. Influence of clinical status on the association between plasma total and unbound bilirubin and death or adverse neurodevelopmental outcomes in extremely low birth weight infants. Acta Paediatr 99: 673-678, 2010]. This suggests that using total bilirubin for determining when to deliver phototherapy may have been misleading because the levels of total bilirubin were not coupled to unbound bilirubin, the toxic fraction of bilirubin. Decoupling of total bilirubin and unbound bilirubin may result from the presence of molecules that interfere significantly with bilirubin binding to albumin. For example, even if total bilirubin was as low as 1 mg/dL, displacement of just 0.2% of total bilirubin by interfering molecules would result in unbound bilirubin=34 nM. This is an unbound bilirubin level that exceeds that thought to be toxic for term newborns and it is generally thought that much lower unbound bilirubin levels would be toxic for premature infants such as those in the Morris et al trial [Morris B H, Oh W, Tyson J E, Stevenson D K, Phelps D L, O'Shea T M, McDavid G E, Perritt R L, Van Meurs K P, Vohr B R, Grisby C, Yao Q, Pedroza C, Das A, Poole W K, Carlo W A, Duara S, Laptook A R, Salhab W A, Shankaran S, Poindexter B B, Fanaroff A A, Walsh M C, Rasmussen M R, Stoll B J, Cotten C M, Donovan E F, Ehrenkranz R A, Guillet R and Higgins R D. Aggressive vs. conservative phototherapy for infants with extremely low birth weight. N Engl J Med 359: 1885-1896, 2008].
Many drugs and metabolites can bind to albumin and as a result, bilirubin is displaced from its bound state on albumin and thereby the unbound concentration of bilirubin is increased whether or not the total bilirubin concentration increases [Spear M L, Stahl G E, Paul M H, Egler J M, Pereira G R and Polin R A. The effect of 15-hour fat infusions of varying dosage on bilirubin binding to albumin. JPEN J Parenter Enteral Nutr 9: 144-147, 1985; Amin S B. Effect of free fatty acids on bilirubin-albumin binding affinity and unbound bilirubin in premature infants. JPEN J Parenter Enteral Nutr 34: 414-420, 2010]. Especially important bilirubin displacing metabolites are free fatty acids (FFA). FFA are always present but are maintained at low levels and do not have a significant effect on healthy term newborns. However under conditions of stress, as for example due to sepsis, FFA levels can increases significantly [Nogueira A C, Kawabata V, Biselli P, Lins M H, Valeri C, Seckler M, Hoshino W, Junior L G, Bernik M M, de Andrade Machado J B, Martinez M B, Lotufo P A, Caldini E G, Martins E, Curi R and Soriano F G. Changes in plasma free fatty acid levels in septic patients are associated with cardiac damage and reduction in heart rate variability. Shock 29: 342-348, 2008]. In addition to disease and stress preterm infants in the NICU can produce extremely large increases in FFA levels as a consequence of receiving by parenteral nutrition an oil emulsion such as Intralipid® [Spear M L, Stahl G E, Paul M H, Egler J M, Pereira G R and Polin R A. The effect of 15-hour fat infusions of varying dosage on bilirubin binding to albumin. JPEN J Parenter Enteral Nutr 9: 144-147, 1985; Amin S B. Effect of free fatty acids on bilirubin-albumin binding affinity and unbound bilirubin in premature infants. JPEN J Parenter Enteral Nutr 34: 414-420, 2010]. FFA bind albumin with high affinities similar to bilirubin. Unlike bilirubin, FFA have multiple high affinity binding sites so that only when an appreciable fraction of the albumin binding sites are occupied by FFA does bilirubin displacement become significant [Spear M L, Stahl G E, Paul M H, Egler J M, Pereira G R and Polin R A. The effect of 15-hour fat infusions of varying dosage on bilirubin binding to albumin. JPEN J Parenter Enteral Nutr 9: 144-147, 1985; Amin S B. Effect of free fatty acids on bilirubin-albumin binding affinity and unbound bilirubin in premature infants. JPEN J Parenter Enteral Nutr 34: 414-420, 2010]. Which newborns receiving Intralipid® will produce large enough quantities of FFA cannot be easily predicted because it depends on gestational age and likely on factors such as enzymatic activity, adiposity and others [Spear M L, Stahl G E, Paul M H, Egler J M, Pereira G R and Polin R A. The effect of 15-hour fat infusions of varying dosage on bilirubin binding to albumin. JPEN J Parenter Enteral Nutr 9: 144-147, 1985; Amin S B. Effect of free fatty acids on bilirubin-albumin binding affinity and unbound bilirubin in premature infants. JPEN J Parenter Enteral Nutr 34: 414-420, 2010]. It is critical to monitor the unbound concentration of FFA (FFAu) during lipid infusion because elevated FFAu levels can cause immune suppression, cardiac damage and reduction in heart rate variability, and elevated levels of unbound bilirubin. In addition, because the unbound levels of these metabolites are dependent upon many patient-specific factors, only by directly monitoring unbound bilirubin during Intralipid® infusion can those infants at risk for bilirubin neurotoxicity be identified. This is particularly true for bilirubin because the elevated plasma levels of FFA caused by increasing Intralipid® concentrations produce elevated unbound bilirubin concentrations without changing the total bilirubin concentration.
Intracellular lipid binding proteins (iLBP) are a family of low-molecular weight single chain polypeptides. There are four recognized subfamilies. Subfamily I contains proteins specific for vitamin A derivatives such as retinoic acid and retinol. Subfamily II contains proteins with specificities for bile acids, eiconsanoids, and heme. Subfamily III contains intestinal type fatty acid binding proteins (FABPs) and Subfamily IV contains all other types of fatty acid binding protein [Haunerland N H and Spener F. Fatty acid-binding proteins—insights from genetic manipulations. Prog Lipid Res 43: 328-349, 2004] including an FABP that binds bilirubin with low affinity [Di Pietro S M and Santome J A. Isolation, characterization and binding properties of two rat liver fatty acid-binding protein isoforms. Biochim Biophys Acta 1478: 186-200, 2000]. The entire family is characterized by a common 3-dimensional fold. Ligand binding properties of the different subfamilies overlap considerably. The wild type proteins of subfamily I [Richieri G V, Ogata R T, Zimmerman A W, Veerkamp J H and Kleinfeld A M. Fatty acid binding proteins from different tissues show distinct patterns of fatty acid interactions. Biochemistry 39: 7197-7204, 2000] and subfamily II both bind fatty acids as well as their native ligands. Moreover, single amino acid substitutions are able to interconvert the ligand binding properties of proteins of subfamilies I and II [Jakoby M G, Miller K R, Toner J J, Bauman A, Cheng. L, Li E and Cistola D P. Ligand-protein electrostatic interactions govern the specificity of retinol- and fatty acid-binding proteins. Biochemistry 32: 872-878, 1993].
U.S. Pat. No. 5,470,714, U.S. Pat. No. 6,444,432, U.S. Pat. No. 7,601,510 and U.S. publication 2010/0298162 which are incorporated herein by reference, describe methods for generating probes and the probes for the determination of unbound analytes. These probes were constructed using either native or mutant forms of proteins from the iLBP family. As discussed above, this family includes FABPs [Banaszak L, Winter N, Xu Z, Bernlohr D A, Cowan S and Jones T A. Lipid-binding proteins A family of fatty acid and retinoid transport proteins. Adv Protein Chem 45: 89-151, 1994; Bernlohr D A, Simpson M A, Hertzel A V and Banaszak L J. Intracellular lipid-binding proteins and their genes. Annu Rev Nutr 17: 277-303, 1997]. FABPs are intracellular proteins of approximately 15 kDa molecular weight and have a binding site that in the wild type proteins binds 1 or 2 FFA as well as other metabolites.